High-pressure, high-resolution liquid chromatography has come into wide use to separate proteins, nucleic acids, metabolites, drugs, and a wide variety of compounds both in research laboratory, in industry, and in clinical laboratories. The systems now in use are expensive, and include non-pulsating high-pressure pumps, valving (often operating at high pressure) for sample introduction, high-pressure prepacked columns, small uniform beads adapted to achieve high resolution separations, a spectrophotometer or colorimeter to monitor each column, and a fraction collector for each column as essential components. In addition, microprocessor systems for analysis of the chromatographs, programs to integrate peaks, and a printer to print out quantitative results are also used. The systems do not include positive means to prevent anomalous flow or channelling to insure ideal flow through the column, to positively remove air bubbles, to prevent a column from running dry during use, to prevent anomalous flow and mixing either in the headspace above the column where flow fans out from the narrow-bore inlet tubing to the full bore of the separations column, or where the converse restriction in diameter occurs as liquid leaves the column and is constricted into the detector flow cell. In addition, the rapid data processing capabilities of microprocessors are not fully used; although one microprocessor can now process, manage, and display data from a dozen or more columns, microprocessors are not so used.
In presently available systems the entire collection of components service, drive, and monitor only one column at a time, can perform only one analysis at a time, is expensive, is subject to a variety of operational problems which may produce anomalous results, and produces resolution lower than that which should, theoretically, be obtained. Additionally, packing new columns or repacking with cleaned column separation materials is difficult if not impossible in the field.
One recent development has altered the instrumental requirements for high resolution separations. This is the development of very uniform spherical beads or resins which reduce the back pressure required to achieve high resolution. Where thousands of pounds per square inch of pressure have been required previously, only hundreds are required now. This means that the pressure requirements can be met in a centrifugal field at much lower speeds. The mechanical strength of the beads has also increased to prevent their deformation under pressure. This higher strength also produces resistance to deformation in centrifugal fields.
Prior noncentrifugal chromatographs encountered numerous other problems. There was no automatic compensation for flow resistance in different columns. When a number of parallel columns are fed from one pump in a non-centrifugal system, the flow through different columns will be slightly different depending on differences in flow resistance in individual columns.
In column chromatography there has not previously been a method for preventing anomalous flow such as channeling through the packing. If the packing is uneven, or if the packing particles are of different sizes, the resistance to flow in different parts of the column will be different. Liquid will flow through the lines or channels of least resistance thereby creating uneven flow. The flow distortions and channeling decrease resolution. Where there are many small anomalies, band widening is observed. Where the anomalies are large, band tilting occurs. Sharp bands which are tilted are also observed as broadened peaks during elution. Extraordinary efforts have gone into the production of column packings of spherical particles of uniform diameter to minimize microanomalous flow. The vastly improved resolution obtained with homogeneous, uniform, particle-sized beads demonstrates the key importance of micro-flow control and minimization of channeling. However, homogeneous particles do not positively prevent anomalous flow. Even with homogeneous particles, the packing may be uneven with local particle compression, or clogging due to the formation of precipitates in the packing may occur. Both conditions produce anomalous flow.
Resolution is also lost during flow through tubing into the column (laminar mixing), during radial flow expansion as the fluid flows from the small bore line leading into the column to the wide bore column, during the decrease in cross-sectional area at the bottom of the column, and by laminar mixing during flow to and in the optical flow cell and the intervening tubing.
Arranging a constant column path length was also difficult. The fluid flowing through the chromatographic system may be considered as being composed of many small fluid elements. In free fluid flow these may be kept in order by centrifugal force acting on a density gradient. However, the solutes in these fluid elements are retarded to different degrees by flow through the separative column packings, and if the path length through the packing is different for different solute elements, considerable loss of resolution occurs. If there is a free space at the ends of the columns, considerable mixing occurs in the absence of a gradient combined with a centrifugal field.
If there is no free space at the ends of the columns and the columns merely constrict to a small diameter at each end, then fluid flowing along the edge of the column will flow through a longer path than does fluid flowing along the center axis of the column, resulting in loss of resolution.
Finally, in conventional chromatography careful attention must be paid to eliminating air bubbles and preventing their formation. In some instances degassing of eluting solutions is required.
These were just some of the problems encountered by prior chromatographs. These, and other problems have been addressed by the centrifugal fast chromatograph of the present invention.